Paclitaxel (Taxol') is mainly produced by species of Taxus. As one of the important achievements in anticancer drug research of 1990's, it has attracted worldwide attention since its advent because of its unique anti-tumor mechanism and prominent anti-tumor activities (Kingston DGI, et al. The taxane diterpenoids. In: Herz W, et al. eds. Progress in the chemistry of organic natural products. New York: Springer-Verlag, 1993, 161-165). It can bind to tubulins, promote the polymerization of tubulins and inhibit the depolymerization thereof, and then hinder the formation of the spindles during the mitosis of cells, such that the cells are stalled in the G2/M phase (Horwitz S B. Taxol (paclitaxel): mechanisms of action. Ann Oncol. 1994, 5 Suppl.). Currently the paclitaxel has been clinically used as a first-line drug for the treatment of breast, ovarian and non-small cell lung cancers, etc. It is also effective against head and neck cancers, melanoma, colon cancer and HIV-induced Kaposi's sarcoma.
The content of paclitaxel in Taxus plants is extremely low and it is mainly present in the bark, which portion has the highest content of paclitaxel, at only about 0.02% (U.S. Pat. No. 6,028,206). A 100-year-old Taxus tree might yield 3 kg of bark which may provide about 300 mg paclitaxel (Horwitz, S B. How to make taxol from scratch. Nature 1994, 367: 593-594). Thus, to harvest 1 kg of paclitaxel from barks needs about 3,000 trees, and 3˜4 one-hundred year old trees are cut down to meet the requirement of one patient's dose. In an alternative method, 10-deacetylbaccatin III with a higher content (up to about 0.1%) is extracted from the leaves of Taxus baccata L., etc, and used as the material for semi-synthesis of paclitaxel and its structural analog, taxotere, which is slightly more active and more soluble in water than paclitaxel (Denis J N, et al. A highly efficient, practical approach to natural taxol. J Am Chem Soc. 1988, 110(17):5917-5919; Horwitz RI. Studies with RP 56976 (Taxotere): A semisynthetic analogue of taxol. J Nat Cancer Inst. 1991, 83(4):288-291; U.S. Pat. No. 4,814,470). The nursery culture of the shrub yew hybrid species is also believed to be the simplest, renewable and the lowest cost way to obtain paclitaxel.
In addition to the very little content of paclitaxel, C-7 xylosyltaxane compounds (taxane-xyloside) having a mother nucleus structure of paclitaxel, including 7-beta-xylosyl-10-deacytyltaxol (XDT), 7-beta-xylosyl-10-deacetylcephalomannine (XDC), and 7-beta-xylosyl-10-deacytyltaxol C (XDTC), etc, have been isolated from yew bark, wherein 7-beta-xylosyl-10-deacytyltaxol (XDT) is most abundant (Senilh V, et al. Mise en evidence de nouveaux analogues du taxol extraits de Taxus baccata. J Nat Prod. 1984, 47:131-137; Rao K V. Taxol and related taxanes. I. Taxanes of Taxus brevifolia bark. Pharm Res. 1993, 10:521-524). For example, XDT, XDC and XDTC could be obtained with the yields of 0.5%, 0.02% and 0.0075%, respectively (EP patent 0,905,130B1). These 7-xylosyltaxane compounds can be hydrolyzed by chemical approaches (U.S. Pat. No. 6,028,206; EP patent 1,298,128B1) or biological approaches (U.S. Pat. No. 5,700,669; CN patent No. 200610046296.6; CN patent No. 200710012698.9) to remove the xylosyl group and generate the corresponding 7-hydroxyltaxanes which can be used in chemical semi-synthesis of paclitaxel or taxotere to enhance the utilization of the source of yew trees and alleviate the imbalance between supply and demand of paclitaxel or its analogues. Comparatively speaking, the chemical approach has some disadvantages, such as relatively low yield, more complicated reaction process and environmental pollution, while the biological approach is more environmentally friendly.
U.S. Pat. No. 5,700,669A, EP patent 0,668,360B1 and relevant articles (Hanson R L, et al. Enzymatic hydrolysis of 7-xylosyltaxanes by xylosidase from Moraxella sp. Biotechnol Appl Biochem 1997, 26: 153-158) disclosed the hydrolyzing method by use of the bacteria Moraxella sp. (ATCC55475), Bacillus macerans (ATCC55476), Bacillus circulans (ATCC55477) and Micrococcus sp. (ATCC55478) to convert C-7 xylosyltaxanes into C-7 hydroxyltaxanes, among which the Moraxella sp. strain showed the highest conversion ability. Adding 0.5 mg 7-xylosyl-10-deacytyltaxol (XDT) to 2 ml of cell suspension (wet cells, 91.5 mg/ml; XDT, 0.25 mg/ml), the suspension was mixed end-over-end at 12 rpm for 21 h at 28° C. The reaction was then stopped with methanol and the sample was assayed by HPLC. No XDT was found to be remaining and the yield of 10-deacytyltaxol (DT) was 0.23 mg/ml.
CN patent (No. 200610046296.6) and relevant article (Hao D C, et al. Bacterial diversity of Taxus rhizosphere: culture-independent and culture-dependent approaches. FEMS Microbiol Lett 2008, 284:204-212) disclosed a hydrolyzing method to convert C-7 xylosyltaxanes to C-7 hydroxyltaxanes using Leifsonia shinshuensis DICP 16 (CCTCC No. M 206026). Similar culturing and converting conditions as those described in above-mentioned US patent is adopted, and 1 mg XDT was added to 2 ml cell suspension. After reacting at 100 rpm for 21 h at 30° C., the reaction was terminated with 2 ml methanol. No XDT was found to be remaining in the reaction solution by HPLC and 0.4 mg/ml DT was produced. In another experiment, different concentrations of 7-xylosyl-10-deacetylbaccatin III (0.5, 0.9, 1.95, 3.1, 4.4, 5.2, and 6.75 mg/ml) were respectively added into 2 ml reaction solution in which the concentration of wet cells was 231.58 mg. The reaction was conducted at 31° C. and 120 rpm for over 40 h. The yield of 10-deacetylbaccatin III reached its highest yield when the concentration of the substrate is 1.95 mg/ml (Hao DC, et al. Bacterial diversity of Taxus rhizosphere: culture-independent and culture-dependent approaches. FEMS Microbiol Lett 2008, 284:204-212).
Another CN patent No. 200710012698.9 disclosed the actinomycete strain Cellulosimicrobium cellulans (XZ-5CCTCC No. M 207130), the hydrolase and their use in the conversion of taxanes: 10 ml XDT (with a concentration of 5 mg/ml) was added into 90 ml crude enzyme solution (1 ml of the Cellulosimicrobium cellulans seed solution cultured at 30° C. for 2 days was introduced into 100 ml medium and cultured at 30° C., 150 rpm for 5 days, and the resultant was centrifuged and the supernatant was isolated to yield the so-called crude enzyme solution) and the reaction was conducted at 30° C. at 50 rpm for 20 h to yield 40 mg DT.
Overall, all the biological approaches mentioned above have potential application values in the hydrolysis of 7-xylosyltaxanes. However, the yields are not high enough to meet the requirement of the industrial mass production, due to the ubiquitous low amount of enzyme in the cells and low substrate solubility in water in the prior arts.
Several kinds of β-xylosidases have been isolated from fungi and other organisms (Tuohy M G, et al. The xylan-degrading enzyme system of Talaromyces emersonii: novel enzymes with activity against aryl beta-D-xylosides and unsubstituted xylans. Biochem J. 1993, 290 (Pt 2):515-523; Golubev A M, et al. Purification, crystallization and preliminary X-ray study of β-xylosidase from Trichoderma reesei. Acta Crystallogr D Biol Crystallogr. 2000, 56 (Pt 8):1058-1060; Pan I, et al. Effective extraction and purification of beta-xylosidase from Trichoderma koningii fermentation culture by aqueous two-phase partitioning. Enzyme Microb Technol. 2001, 28 (2-3):196-201; Rizzatti A C S, et al. Purification and properties of a thermostable extracellular β-D-xylosidase produced by a thermotolerant Aspergillus phoenicis. J Ind Microbiol Biotechnol. 2001, 26(3):156-160; Saha B C. Purification and characterization of an extracellular β-xylosidase from a newly isolated Fusarium verticillioides. J Ind Microbiol Biotechnol. 2001, 27 (4):241-245; Gargouri M, et al. Fungus beta-glycosidases: immobilization and use in alkyl-beta-glycoside synthesis. J Mol Catal B: Enzym. 2004, 29, Issues 1-6:89-94; Lama L, et al. Purification and characterization of thermostable xylanase and β-xylosidase by the thermophilic bacterium Bacillus thermantarcticus. Res Microbiol. 2004, 155(4):283-289; Belfaquih N & Penninckx M J. A bifunctional β-xylosidase-xylose isomerase from Streptomyces sp. EC 10. Enzyme Microb Technol. 2000, 27(1-2): 114-121), and some β-xylosidase genes (such as those from several fungus sources) have been cloned and identified successfully (Margolles-Clark E, et al. Cloning of genes encoding alpha-L-arabinofuranosidase and beta-xylosidase from Trichoderma reesei by expression in Saccharomyces cerevisiae. Appl Environ Microbiol. 1996, 62(10):3840-3846.; van Peij N N, et al. β-Xylosidase activity, encoded by xlnD, is essential for complete hydrolysis of xylan by Aspergillus niger but not for induction of the xylanolytic enzyme spectrum. Eur J Biochem. 1997, 245 (1):164-173; Perez-Gonzalez J A, et al. Molecular cloning and transcriptional regulation of the Aspergillus nidulans xlnD gene encoding a β-xylosidase. Appl Environ Microbiol. 1998, 64(4):1412-1419; Kitamoto N, et al. Sequence analysis, overexpression, and antisense inhibition of a β-xylosidase gene, xylA, from Aspergillus oryzae KBN616. Appl Environ Microbiol. 1999, 65(1):20-24; Berrin J G, et al. High-level production of recombinant fungal endo-β-1,4-xylanase in the methylotrophic yeast Pichia pastoris. Protein Expr Purif. 2000, 19(1): 179-187; Reen F J, et al. Molecular characterisation and expression analysis of the first hemicellulase gene (bxl1) encoding β-xylosidase from the thermophilic fungus Talaromyces emersonii. Biochem Biophys Res Commun. 2003, 305(3):579-585; Kurakake M, et al. Characteristics of transxylosylation by beta-xylosidase from Aspergillus awamori K4. Biochim Biophys Acta. 2005, 1726(3):272-279; Wakiyama M, et al. Purification and properties of an extracellular β-Xylosidase from Aspergillus japonicus and sequence analysis of the encoding gene. J Biosci Bioeng. 2008, 106(4):398-404). However, none of these (natural or recombinant) β-xylosidases was found to have the ability of specifically hydrolyzing 7-xylosyltaxanes. Therefore it is reasonably believed that the genes of β-xylosidases with specific catalytic activity against 7-xylosyltaxane compounds have not been cloned so far, not to mention functional analysis. In fact, a lot of commercial xylosidases, xylanases and other glycosidase did not reveal the ability to remove the xylosyl group from 7-xylosyltaxanes at all (Hanson R L, et al. Enzymatic hydrolysis of 7-xylosyltaxanes by xylosidase from Moraxella sp. Biotechnol Appl Biochem 1997, 26: 153-158).